Osteoblast differentiation and bone formation are controlled by a wide range of extracellular signals including cell-extracellular matrix interaction, matrix stiffness, biomechanical force, hormones, growth factors and morphogens. To induce differentiation, each stimulus must initiate a signal that can be efficiently dispersed through the genome to induce global changes in gene expression necessary for the osteoblastogenesis while simultaneously suppressing other lineages. The following hypothesis is proposed to explain how this is accomplished: i) After exposure to an osteogenic stimulus, P-ERK1/2 translocates to the nucleus and binds chromatin of osteoblast target genes via specific docking sites on Runx2. ii) By phosphorylating RUNX2 on specific sites, kinases initiate chromatin remodeling and transcription; the overall level of RUNX2 phosphorylation is a reflection of the various extracellular stimuli to which the cell is exposed. iii) Conversely, kinases may phosphorylate other lineage-specific transcription factors on chromatin to suppress non-osteogenic lineages such as adipocytes. This hypothesis will be tested by achieving the following aims: 1. Evaluate the role of MAP kinase phosphorylation of RUNX2 and PPAR? in the reciprocal control of osteogenesis and adipogenesis. 2. Evaluate the role of RUNX2 phosphorylation by ERK1/2 in the control of fluid flow shear stress (FFSS)-induced osteoblast gene expression. 3. Determine the role of Runx2 S301,S319 phosphorylation in skeletal development and remodeling. Experiments include detailed epigenetic analyses of chromatin changes accompanying osteoblast versus adipocyte differentiation as well as genomic analysis using chromatin immunoprecipitation and next generation sequencing. These studies will test a new model for global control of gene expression that may explain how osteogenic stimuli, including extracellular matrix signals and mechanical loading, can reprogram the genome to stimulate osteoblast differentiation while suppressing other lineages such as adipocytes. Once this model is understood, it will be possible to manipulate osteogenic signals to optimize bone formation during remodeling and regeneration. Furthermore, this work will test the validity of using RUNX2 phosphorylation-site specific antibodies to monitor activation of this factor under different physiological conditions. This may have applications for diagnosis of osteoporosis and other bone disorders, such as osteoarthritis and certain cancers.